Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X-Ray Imaging

The iridium half-sandwich complex [Ir(η⁵:κ¹-C₅Me₄CH₂py)(2-phenylpyridine)]PF₆ is highly cytotoxic: 15–250× more potent than clinically used cisplatin in several cancer cell lines. We have developed a correlative 3D cryo X-ray imaging approach to specifically localize and quantify iridium within the whole hydrated cell at nanometer resolution. By means of cryo soft X-ray tomography (cryo-SXT), which provides the cellular ultrastructure at 50 nm resolution, and cryo hard X-ray fluorescence tomography (cryo-XRF), which provides the elemental sensitivity with a 70 nm step size, we have located the iridium anticancer agent exclusively in the mitochondria. Our methodology provides unique information on the intracellular fate of the metallodrug, without chemical fixation, labeling, or mechanical manipulation of the cells. This cryo-3D correlative imaging method can be applied to a number of biochemical processes for specific elemental localization within the native cellular landscape.     

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.     

Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures

Techniques based on two-photon excitation (TPE) allow three-dimensional (3D) imaging in highly localized volumes, of the order of magnitude of a fraction of a femtolitre up to single-molecule detection. In TPE microscopy a fundamental advantage over conventional widefield or confocal 3D fluorescence microscopy is given by the use of infrared (IR) instead of ultraviolet (UV) radiation to excite those fluorophores requiring UV excitation, hence causing little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sample compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realized a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture is a mode-locked Ti:sapphire infrared pulsed laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse width from 65 to 95 fs as a function of wavelength (690-830 nm). The scanning head allows conventional and two-photon confocal imaging. Point spread function measurements are reported with examples of applications to the study of biological systems.     

Use of 3D mesh geometries and additive manufacturing in neutron beam experiments

PGAI-NT, a neutron-based element composition and structure analysis method, is well applicable to the non-destructive characterization of valuable artefacts, paleontological, bulk geological samples or to industrial reverse engineering. To set up the measurement geometry and scanning positions for items with unconstrained shapes, sizes, and matrices, accurate knowledge of the object’s geometry is a must. We applied portable structured-light 3D optical scanning or segmented neutron/X-ray tomography data to produce 3D meshes of the complex samples. Subsequently, 3D printing was used to fabricate detailed replicas of museum objects, as well as their gentle ad hoc sample holders, or produce custom parts of the Budapest PGAA instrument.

     

X-ray fluorescence imaging with polycapillary X-ray optics

X-ray fluorescence spectrometry imaging is a powerful tool to provide information about the chemical composition and elemental distribution of a specimen. X-ray fluorescence spectrometry images were conventionally obtained by using a μ-X-ray fluorescence spectrometry spectrometer, which requires scanning a sample. Faster X-ray fluorescence spectrometry imaging would be achieved by eliminating the process of sample scanning. Thus, we developed an X-ray fluorescence spectrometry imaging instrument without sample scanning by using polycapillary X-ray optics, which had energy filter characteristics caused by the energy dependence of the total reflection phenomenon. In the present paper, we show that two independent straight polycapillary X-ray optics could be used as an energy filter of X-rays for X-ray fluorescence. Only low energy X-rays were detected when the angle between the two optical axes was increased slightly. Energy-selective X-ray fluorescence spectrometry images with projection mode were taken by using an X-ray CCD camera equipped with two polycapillary optics. It was shown that Fe Kα (6.40 keV) and Cu Kα (8.04 keV) could be discriminated for Fe and Cu foils.     

Waveguide confined Raman spectroscopy for microfluidic interrogation

We report the first implementation of the fiber based microfluidic Raman spectroscopic detection scheme, which can be scaled down to micrometre dimensions, allowing it to be combined with other microfluidic functional devices. This novel Raman spectroscopic detection scheme, which we termed as Waveguide Confined Raman Spectroscopy (WCRS), is achieved through embedding fibers on-chip in a geometry that confines the Raman excitation and collection region which ensures maximum Raman signal collection. This results in a microfluidic chip with completely alignment-free Raman spectroscopic detection scheme, which does not give any background from the substrate of the chip. These features allow a WCRS based microfluidic chip to be fabricated in polydimethylsiloxane (PDMS) which is a relatively cheap material but has inherent Raman signatures in fingerprint region. The effects of length, collection angle, and fiber core size on the collection efficiency and fluorescence background of WCRS were investigated. The ability of the device to predict the concentration was studied using urea as a model analyte. A major advantage of WCRS is its scalability that allows it to be combined with many existing microfluidic functional devices. The applicability of WCRS is demonstrated through two microfluidic applications: reaction monitoring in a microreactor and detection of analyte in a microdroplet based microfluidic system. The WCRS approach may lead to wider use of Raman spectroscopy based detection in microfluidics, and the development of portable, alignment-free microfluidic devices.     

Theory of the implementation of the fundamental parameter method for the X-ray fluorescence determination of low-atomic-number elements

The theory of X-ray fluorescence generation in elements with a low atomic number Z is extended to the case of host matrices with high Z. The total contribution of the selective excitation effects is estimated with regard to the cascade K-L transitions. The influence of the elemental composition of the matrix and the excitation conditions on the X-ray fluorescence intensity of the elements in study is assessed. The accuracy of the model of X-ray fluorescence generation in the low-atomic-number elements is confirmed by the agreement of the experimental and calculated intensities of carbon in various carbon-containing compounds.     

Error reduction in simultaneous proton induced X-ray emission tomography and on/off-axis scanning transmission ion microscopy-tomography

Rapid micron-resolution quantitative elemental mapping is possible at the University of Surrey using a combination of proton induced X-ray emission tomography (PIXE-T) and simultaneous on/off-axis scanning transmission ion microscopy-tomography (STIM-T). A preliminary analysis of hair was performed. However, experimental uncertainties lead to large errors in tomograms and this work focuses on identifying and reducing the sources of error in both tomographic and 2D mapping. The STIM-T counts per pixel are used to normalise the PIXE-T data for charge. However, the geometry of the collimator and the scattering foil affects the detection rate since the loss of protons in the collimator increases as energy loss increases due to scattering. Errors in the PIXE geometric efficiency are greater in mapping when the detector is close to the sample. Moreover when a ‘funny’ filter was used for PIXE-T the uncertainty in the efficiency was found to increase because the sample-filter distance changes during the experiment.     

Two novel rhodamine-perylenediimide fluorescent probes: Synthesis,photophysical properties,and cell imaging

In this study, two novel dual-switch fluorescent chemosensors based on rhodamine-peryleneiimide have been designed and synthesized. The dual-switching behaviors of the sensors were based on the structural transformations of rhodamine and an intramolecular photoinduced electron transfer (PET) process from rhodamine to perylenediimide. These probes exhibited excellent sensitivity to protons with enhanced fluorescence emission from 500 nm to 580 nm. The fluorescence changes of probes were reversible within a wide range of pH values from 2.0 to 11.0. Moreover, the sensors exhibited high selectivity, short response time, and long lifetime toward protons. The possible mechanism was investigated by the DFT calculation and 1 H NMR. According to the experiment of confocal laser scanning microscopy, these probes could be used to detect the acidic pH variations in living cells.     

Study of a XVIII century hand-painted Chinese wallpaper by multianalytical non-destructive techniques

In this work, hand-painted wallpaper belonging to a private Portuguese collection was analyzed in order to identify the pigments used. The analyzed artwork was an extraordinary XVIII century Chinese wallpaper that depicts exotic birds and flowers, which was painted with considerable accuracy and expertise. Thorough, in situ, X-ray fluorescence analyses were performed on nearly all the wallpaper. Since the elemental content of several colors was consistent for the four papered walls, strategic micro-samples were taken and analyzed by confocal Raman spectroscopy to further identify the pigments used. Pigments such as yellow ochre, lead white and barium white, vermilion, carmine, azurite and malachite were identified. Optical microscopy was used to analyze the fibers in the paper support, and fibers such as kozo, ramie and hemp or linen were identified.     

Rapid, photoactivatable turn-on fluorescent probes based on an intramolecular photoclick reaction

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-"uncaging" and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction ("photoclick chemistry"). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (～1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid-tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid-tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.     

EPMA Measurements of Diffusion Profiles at the Submicrometre Scale

 Concentration profiles due to (inter)diffusion in materials may require high spatial resolution. These profiles may be measured by electron probe microanalysis, which allows one to determine the elemental composition with a good accuracy provided measurement ‘artefacts’ can be accounted for. Standard phenomena are usually corrected by commercial softwares that assume a homogeneous elemental composition in the analysed area. However, in the case of a diffusion process on a small scale, the composition is no longer homogeneous and the effect of the hemispherical volume of the X-ray emission on the spatial resolution of the concentration profiles, and consequently on the diffusion coefficients, has to be considered. Moreover, (secondary) fluorescence across interfaces or interphases has to be evaluated. A radial X-ray distribution associated with the characteristic depth distribution, φ(ρz), allows for the definition of a 2D X-ray emission function that enables the computation of the entire process for a given concentration profile.     

X-ray fluorescent analysis using synchrotron radiation: Subjects of research

The brief review has been presented about the application of X-ray fluorescent analysis using synchrotron radiation (storage ring VEPP-3, BINP SB RAS) for determination of elemental composition of the samples of different nature–biological and geological samples, objects of environment, archeological objects, and new materials. The feature of the presented research is the employment of the unique properties of synchrotron radiation, which allow analyzing samples of small mass (of the order of several milligrams), and also scanning core of bottom sediments with high resolution (less than 1 mm) that is not practical to realize by traditional analysis methods.     

Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging

The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.     

Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein–nanomaterial hybrids

Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein–nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine.

A novel 3D cryo correlative approach locates designed therapeutic protein–nanomaterial hybrids in whole cells with high specificity and resolution. Detection of treatment-induced morphological changes, crucial for pre-clinical studies, are revealed.     

双光子荧光有机纳米粒子的细胞染色

本文采用具有较大双光子吸收截面的有机分子2,5,2′,5′-(4′-N,N-二苯胺苯乙烯基)联苯(DPA-TSB)(双光子吸收截面δ: 3288 GM, 1 GM=1×10^-50 cm⁴·s·photon^-1·molecule^-1), 通过再沉淀法制备水相分散的纳米粒子. 研究表明, 这种有机双光子纳米粒子可以有效地富集在细胞质中, 对细胞染色显示出良好的荧光成像能力.     

Imaging methods for elemental, chemical, molecular, and morphological analyses of single cells

Combining elemental, chemical, molecular, and morphological imaging information from individual cells with a lateral resolution well below 1?×?1 μm² is the current technological challenge for investigating the smallest dimensions of living systems. In the race for such analytical performance, several techniques have been successfully developed; some use probes to determine given cellular contents whereas others use possible interactions between cellular matter with light or elements for characterization of contents. Morphological techniques providing information about cell dimensions have, when combined with other techniques, also opened the way to quantitative studies. New analytical opportunities are now being considered in cell biology, combining top-performance imaging techniques, applied to the same biosystem, with microscopy (nm–μm range) techniques providing elemental (micro-X-ray fluorescence, particle-induced X-ray emission, secondary-ion mass spectrometry), chemical (Raman, coherent anti-stokes Raman, Fourier-transform infrared, and near-field), molecular (UV–visible confocal and multiphoton), and morphological (AFM, ellipsometry, X-ray phase contrast, digital holography) information. Dedicated cell-culture methods have been proposed for multimodal imaging in vitro and/or ex vivo. This review shows that in addition to UV–fluorescent techniques, the imaging modalities able to provide interesting information about a cell, with high spatial and time resolution, have grown sufficiently to envisage quantitative analysis of chemical species inside subcellular compartments.     

基于迈克尔加成反应的沙门氏菌纳米荧光标记研究

本文通过迈克尔加成反应的荧光标记法,成功构建了纳米荧光探针标记的沙门氏菌(YB1-INPs)。采用三(2-羧乙基)膦将沙门氏菌YB1外膜蛋白表面上的二硫键温和还原为巯基后,与吲哚菁绿(ICG)@纳米探针(INPs)的马来酰亚胺基团(Mal)交联形成YB1-INPs。通过扫描电镜和共聚焦荧光成像对YB1-INPs的形貌、光学性质进行表征。通过活死细菌染色和LB琼脂平板实验对YB1-INPs的活性进行考察。结果表明,INPs成功标记到沙门氏菌YB1表面后对YB1的形态、光学性质、活性及生长均没有产生影响,并且能够用于沙门氏菌YB1体内靶向的荧光分布成像。本研究提供的这种简单、安全、高效的荧光标记方法能够将纳米材料标记到生物载体上,可以应用于生物载体的药物输送和体内监测。     

Confocal microscopic X-ray fluorescence at the HASYLAB microfocus beamline: characteristics and possibilities

The possibilities of performing non-destructive elemental analysis in three dimensions on a variety of heterogeneous materials by means of an innovative variation of the microscopic X-ray fluorescence analysis (μ-XRF) method are described. Next to employing focusing optics for concentration of the primary beam of X-rays, a second optical element between the sample and the energy-dispersive detector is used in confocal μ-XRF. Thus, only X-ray fluorescence signals from a cube-like volume (within certain limits imposed by the absorption of the radiation in the sample) can be arbitrarily positioned within the sample. The distribution of major, minor and trace elements (down to the sub-ppm concentration level in some matrices) along lines and planes within the sample can be visualized with a spatial resolution of the order of 15–40 μm. The lowest detectable amounts in confocal mode using pink-beam excitation are situated at the sub-femtogram level.